The World Health Organization (WHO) supplies reference reagents to its participating laboratories for distribution to researchers. Recently a need was established for reference sera with neutralizing antibody (NA) titers to HIV-1. In response to this request, our laboratory selected serum samples from ten HIV-1 antibody positive patients initially identified at the NIH Blood Bank to be screened for NA titers. Two patients were selected based on medium to high NA titers and were asked to donate a unit of blood on 3-4 occasions over a 9-month period of time. The blood was clotted and the serum was collected and stored at 40C. Samples from each bleed date were tested for NA responses against a few strains of HIV-1. After all of the bleeds, serum from each patient was pooled separately and prediluted 1:4 with PBS and sterile glass-distilled water before they were aliquoted into glass ampules and freeze-dried. The sera were tested after they were reconstituted to be sure there was no change in titer. An international collaborative study was arranged according to the WHO guidelines with 6 laboratories throughout the world (including our own laboratory) being asked to use their own in-house method for determining NA titers. A panel of ten coded sera including the two reference sera were aliquoted and lyophilized in the same manner along with a positive and negative control and sent to each of the participating laboratories. Data from these laboratories will be summarized to determine the value that WHO will assign to these reference reagents. Other required tests to establish these two samples as reference reagents, such as an infectivity assay, a stability study, accuracy of fill volume and moisture content will also be done.